471
J. Phy8&l. (1978), 278, pp. 471-490 With 12 text-figure8 Printed in Great Britain
THE HALF-LIVES OF ANGIOTENSIN II, ANGIOTENSIN II-AMIDE, ANGIOTENSIN III, SAR1-ALA8-ANGIOTENSIN II AND RENIN IN THE CIRCULATORY SYSTEM OF THE RAT
BY S. A. M. A. AL-MERANI, D. P. BROOKS, B. J. CHAPMAN AND K. A. MUNDAY and Pharmacology, School of Biochemical and Physiology From the Department of
Physiological Sciences, University of Southampton, Southampton S09 3TU (Received 3 November 1977) SUMMARY
1. Methods are described for estimating the half-life of angiotensin analogues and renin in the rat, from the time course of the blood pressure changes they evoke. 2. The following half-life values were measured: angiotensin II, 16 + 1 see; angiotensin III, 14+ 1 see; angiotensin II-amide, 15±+1 see; Sarl-Ala8-angiotensin II, 6-4 + 0-6 min; renin, 3 0 + 0 4 min. The apparent distribution volume of angiotensin was found to be 18 ml./kg body wt. 3. It is inferred that the Asp' residue does not reduce the rate of angiotenrsin II catabolism, but that substitution of this residue by sarcosine may inhibit catabolism while substitution by asparagine has no effect. 4. Five experimental criteria were identified which indicate that these methods give reliable estimates of the half-life. It is suggested that these results are more accurate than most previous half-life estimates. 5. When tachyphylaxis to angiotensin II-amide occurs, the pressor activity of the plasma is not reduced. INTRODUCTION
Studies of the half-life (t4) of angiotensin II have produced some conflicting results and very little is known of the half-lives of angiotensin analogues. Hodge, Ng & Vane (1967) measured the capacity of various organs to remove angiotensin II from the circulation, and calculated a t1 of 15 see in the dog. Wolf, Mendlowitz, Gitlow & Naftchi (1962) measured the t1 of [131I]angiotensin II as between 10 and 37 hr in man, although this probably represents the biological half-life of the 131I after break-down of the angiotensin molecule. Corcoran, Kohlstaedt & Page (1941) assessed the tj of a crude angiotensin preparation to be 20-25 min. Cain, Catt, Coughlan & Blair-West (1970), and Boyd, Landon & Peart (1969) found a tj of about 1 min for angiotensin II; but these workers used immunoassay techniques which may have detected inactive peptides formed by angiotensin hydrolysis, and hence may have underestimated the inactivation of the hormone. Regoli, Rioux, Park & Choi (1974) found that Sarl-angiotensin II has a reduced rate of catabolism in vitro and concluded that the greater pressor activity of the molecule may well result from a longer half-life in vivo. Similarly, Peach (1977) has sug-
S. A. M. A. AL-MERANI AND OTHERS gested that the in vivo tj of des-Aspl-angiotensin II may be 2-3 times shorter than that of angiotensin II, since it is catabolized faster in vitro. There are therefore important reasons for determining accurately the half-lives of angiotensin II and related compounds in the circulatory system, especially since it is possible to relate such measurements to studies of the in vivo actions of these substances. We wanted to measure these half-lives in the rat, but the rat is too small to permit the withdrawal of serial blood samples for angiotensin II assay and also there are no assay methods for some angiotensin analogues. We have therefore measured the half-lives of these various substances by studying the time course of the pressor response when a continuous infusion of each substance was initiated or arrested. The half-life of a hormone is usually determined by measuring the decrease in hormone concentration when an infusion of the hormones is stopped. However, it is not essential to measure the hormone concentration in absolute units; it is only required to find the time taken for the concentration, however expressed, to change by a factor of 0.5. It is established that the rise in plasma concentration of angiotensin II is proportional to the rate at which it is infused; any plasma concentration can therefore be quantified in terms of the infusion rate which could generate that concentration. We have assessed the changes in hormone concentration as follows: blood pressure readings were taken at regular intervals and interpreted as 'equivalent infusion rates' by reference to a calibration graph of blood pressure vs. infusion rate. Some of this work has been included in a brief report elsewhere (Brooks, Chapman & Munday, 1977). 472
METHODS
250 g female, Wistar albino rats (given 0-9 % saline in place of their drinking water for at least 10 days) were anaesthetised with sodium pentobarbitone and cannulae were placed in the trachea, one or both femoral veins (for infusion and/or injection of drugs) and in a femoral artery (for the measurement of blood pressure using an S.E. Labs. blood pressure transducer and a Servoscribe, model 45, potentiometric recorder). The following substances were dissolved in 0 9 % saline and either injected i.v. in 0 1-0 2 ml. or infused in 1 ml./hr at a variety of doses: angiotensin II-amide, i.e. Asn.-Valk-angiotensin II (Hypertensin, Ciba), 3-17,000 ng. kg-l. min'; angiotensin II, i.e. Asp'-Ile6-angiotensin II (Digby Chemical Company), 3-1000 ng. kg-'. min'; angiotensin III, i.e. des-Asp'-angiotensin II (D.C.C.), 20-4000 ng.kg-1.min-'; Saralasin, i.e. Sarl-Ala8-angiotensin II (D.C.C.) 5-200jg; Sarl-Leu8-angiotensin II (D.C.C.) 1 ,ug.kg-1.min-1; tyramine (Sigma) 200 jug; noradrenaline (Sigma) 20 jug; and pig renin (very kindly prepared by B. Rowe and A. R. Noble using the method of Haas, Goldblatt, Lewis & Gipson, 1972). The various doses of any chemical were applied in random sequence. Estimation of the ti values. Ginsberg (1968) and Goldstein, Aronow & Kalman (1968) have described the relationship between the infusion rate of a hormone (I), the volume (V) in which the hormone is distributed within the body, the t4, and the plasma concentration of the hormone at equlibrium (C): I~ (1) 0.693. V Since the 4 and V normally remain constant in any one animal, C is proportional to I; this has been verified in the case of angiotensin (see below). Thus any given infusion of angiotensin, I, will give rise to an equilibrium concentration C of the hormone and a pressor response R'. If the hormone is infused at 2 x I then the concentration of hormone at equilibrium will become 2 x C' and this will give rise to the pressor response R'. This pressor response R' will not necessarily be equal to 2 x R'. If this infusion is stopped, the concentration of hormone will decrease exponentially and the pressor response will dissipate at a rate determined by the disappearance rate of the
e=
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES
473
hormone (evidence in support of this is given below). When the pressor response has fallen to R' then the concentration of hormone must have fallen from 2C to C and the time taken must be equal to the half-life of the hormone. This principle was extended by measuring the magnitude of the pressor response at 5 see intervals after stopping the infusion. These responses were related to 'equivalent infusion rates' (and thus related to the concentration of hormone, since this is proportional to an infusion rate) by reference to a graph of equilibrium pressor response verbus infusion rate. Graphs of log 'equivalent infusion rate' versus time were constructed and the half lives determined from the slope of the resulting straight lines; ij = log 2/slope. For some experiments this basic method was further simplified by constructing graphs of (the peak pressor response) verseu (log dose) for single injections of the hormones; the time course of the decay of any response was then studied as described above. This is referred to as the simplified injection procedure. Goldstein et al. (1968) have shown that when an infusion of a hormone is started, the concentration of the hormone increases towards the equilibrium (plateau) concentration with a shift halftime that is equal to the half-life of the hormone. In some experiments the time course of the onset of the pressor response was therefore studied. The blood pressure was measured at five second intervals following the start of an infusion and graphs were plotted to show log (the infusion rate minus the 'equivalent infusion rate') versus time. The shift half-time, i.e. the tj of the hormone, was calculated from the slope of the resulting straight line, The half-life of Saralasin, which lacks pressor activity but is a potent inhibitor of angiotensin II action, was investigated as follows. 300 ng. kg-'. min- angiotensin II was continuously infused into each rat to give a constant rise in blood pressure, and various doses of the inhibitor (5200 jog) were injected i.v. in random sequences. The blood pressure falls due to the inhibitor were recorded and calibration graphs were constructed showing depressor response vernue log dose. From the recordings of the return of the blood pressure following the point of maximum inhibitory action of the Saralasin, graphs of log 'equivalent dose' verbu. time were constructed and the half-life of the inhibitor was determined. Bio-a88ay of pre88or activity in blood. Arterial blood samples (0-1 ml.) were taken from rats receiving 0-17-5 jug angiotensin II-amide kg-".min-' and injected I.v. into ganglion-blocked (pentolinium tartrate, 10 mg/kg) bio-assay rats (Peart, 1955). In some cases the pressor activity of these blood samples was assayed in terms of angiotensin II-amide concentration by bracketing with standard doses (Peart, 1955). The measured concentrations of angiotensin II-amide were corrected for catabolism during the period of transfer, by adding known amounts of angiotensin II-amide to blood samples and assaying the resulting pressor activity. An antagonist of angiotensin II pressor activity (Sarl-Leu8-angiotensin II) was infused at 1 #g/min i.v. into some bio-assay rats to give a preparation capable of detecting non-angiotensin pressor activity. All results are quoted as the mean ± 1 S.E. of the mean. The methods described by Colquhoun (1971) were used to evaluate the results statistically.
RESULTS
The tj of angiotensin II-amide Figs. 1 and 2 show the blood pressure recordings and the calibration graph of (plateau pressor response) versus (log infusion rate) obtained from one rat. The infusions were given for 2-5 min and were applied in random sequence but have been edited as shown. The decays of the pressor responses were studied for the three highest doses and used to construct the lines shown in Fig. 3. The average tt of angiotensin II-amide measured in this rat was 150O sec. The t1 of angiotensin II-amide was thus found to be 15-2 + 1-1 sec in sixteen rats. The measured tj was found to be the same for decays following a wide range of infusion rates (Fig. 4). These results show that the pressor responses were able to dissipate at very fast rates following high doses of the drug and that the initial,
474 S. A. M. A. AL-MERANI AND OTHERS steepest rate of decrease of the blood pressure, was proportional to the dose of angiotensin II-amide studied. There was usually a short time delay between switching off the infusion of the hormone and the start of the downsweep of the pressure recording. This delay, which was quantified by extrapolating the linear portion of the decay line as shown in Fig. 3, was found to be 10-4 + 0 7 seconds (n = 15). This delay was generally independent of the dose administered, and may have been due to the time taken for blood to travel between the site of infusion, the femoral vein, and the site of drug action. There may also be a time lay between the disappearance of the hormone and the reaction of the target tissue (see below). Dose of angiotensin lI-amide 3 ng. kg-'. min' 30 30 10
*6)
(mmHg)
-1110
I
_
Rise inB.P.at plateau
Rise= 6 mmHg
100
126
300
-1000
is
____
__
_15
T~~~~~~~~~~~~4
I~~~~~~
Fig. 1. Arterial blood pressure recordings obtained from a typical rat before, during and following various i.v. infusions of angiotensin II-amide. The speed of the chart recorder was increased at the points marked *, to permit detailed study of the decay curves.
The shift half-time was also determined from the onset of the pressor responses produced when various angiotensin II-amide infusions were switched on (Fig. 5). The shift half-time is the time for the concentration of a hormone to move 50% of the way towards its equilibrium concentration, when an infusion is started or changed. Goldstein et al. (1968) have shown theoretically and empirically that the upsweep of the concentration is a mirror-image of the disappearance of hormone when an infusion is switched off. The shift half-time is therefore equal to the tj for the destruction of the hormone. It was only possible to consider the initial part of the rise in blood pressure as the calibration curves did not have sufficient precision to permit evaluation of changes close to the plateau. The initial rate of increase of blood pressure was proportional to the dose of angiotensin II-amide used when the lower doses were infused; but when the two higher doses were used, the rate of increase of blood pressure was no longer proportional to the infusion rate (Fig. 6A). Similarly the
HALF-LIVES OF REIINJN AND ANGIOTENSIN ANALOGUES 475 measured half-life (i.e. the shift half-time) was independent of the infusion rate at the lower doses, but was observed to be prolonged during the infusion of 1000 ng. kg-1. min-' and to be slightly prolonged during the infusion of 500 ng. kg-1. min(see Figs. 5 and 6B). These observations are interpreted as indicating that the concentration of angiotensin II-amide follows a time course with a constant shift halftime, but that the rate of onset of the pressor response is limited by a maximum rate
60r-
40 0
I
E E C
U, .
_
20-
n v
0
100 10 Infusion rate of angiotensin 11 - amide (ng kg-1 min')
1000
Fig. 2. Calibration curve of the rise in B.P. vergu8 the infusion rate of angiotensin IIamide (taken from Fig. 1). The points were measured at the plateaux of the blood pressure responses.
of reaction of the target tissue (e.g. a maximum rate of contraction of the vascular smooth muscle). The results for the lower three doses were averaged for each animal. The over-all mean tj of angiotensin II-amide in six rats was then calculated and found to be 16-3 + 2 1 sec. This value was not significantly different from that calculated for the disappearance of the angiotensin II-amide (P > 0 05, Wilcoxon test). The half-life of angiotensin JI-amide was also determined by the simplified injection procedure (see Methods section) as 15-1 + 1t3 sees (n = 5 rats). This value is very similar to that obtained following constant infusions of the hormone (P > 0.05, Wilcoxon test), which indicates that the simplified injection procedure can give the same half-life results as does stopping a continuous infusion.
.' \t ~ ~ ~Trace6
S. A. M. A. AL-MERANI AND OTHERS
476
The half-life of natural angiotensin II and angiotensin III The half-lives were also determined for the disappearance of various analogues of angiotensin, which also have pressor activity: these were all found to have half-lives very similar to that of angiotensin II-amide (Table 1). 1000
From
Fig. 1
E
\U
6
Time lag
Measured,
t; (sec)
Trace 5
135
\Tae4
1-
100
15 *
30
-
Cr
10 50 10 20 30 40 60 Time after discontinuing angiotensin 11-amide infusion (sec)
Fig. 3. The exponential decrease in angiotensin II-amide concentrations, when various i.v. infusions were discontinued. Examples from one rat, taken from curves 4, 5, and 6 of Fig. 1. See Methods section for an explanation of how these graphs were constructed.
The half-life of rein The half-life of renin was determined by the simplified injection procedure and was found to be 3*02 + 0*41 min in four rats (Table 1). The half-life of Saralasin The half-life of Saralasin was found to be 6-43 ± 0*57 min in six rats; the modified procedure described in the Methods section was used.
Tests of the validity of the methods used. The initial rate of decrease of the blood pressure was determined for a variety of experimental conditions (Figs. 4, 7). When an infusion of 300 ng. kg-. min-' angiotensin JI-amide was discontinued the blood pressure initially decreased at 37*4 +
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES 477 2*6 mmHg. min-1 in seventeen rats (Fig. 8C). When an i.v. infusion of 25 ,ag.kg-1. min- acetylcholine was given to animals receiving an infusion of 300 ng . kg-.. mi r1 angiotensin II-amide, the blood pressure initially decreased at 1470 + 290 mmHg. min- (n = 4 rats) which is significantly greater than the rate of decrease following the arrest of the angiotensin II-amide infusion (P < 0 01, Wilcoxon test). CL 0. O.
100r
A
4,
: E 0)
tcX 0
(5)
E
a
(U' E
(11
00.C CD
E
C
30
0
0a)
Mean tj
0
-
15-2+1-1
0
300 1000 50 100 Dose of angiotensin 11-amide studied (ng kg-'
sec
4000
min-')
Fig. 4A, the initial rate of decrease of blood pressure versus the infusion rate of angiotensin II-amide studied. B, the dependence of the measured half-life on the infusion rate at which the measurement was obtained. (These graphs are plotted on log scales to accommodate the wide range of doses used; the numbers of animals are shown in parentheses.)
The initial rates of decrease of blood pressure were also measured following various 1.v. injections of Saralasin in animals receiving a sustained infusion of 300 ng.kg-'. min- angiotensin II-amide (Fig. 7). The rate of decrease of blood pressure following the highest dose of inhibitor (200 jug) was 70 + 11 mmHg. min' (n = 6 rats). This was significantly faster than the decrease following the arrest of an angiotensin II-amide infusion (P < 0*01, Wilcoxon test). Furthermore, five of the doses of Saralasin caused
478 S. A. M. A. AL-MERANI AND OTHERS the blood pressure to decrease faster than did stopping the angiotensin II-amide infusion (compare Fig. 8B, C). Graphs were plotted of the reciprocal of (the initial rate of decrease in blood pressure) versus the reciprocal of (the dose of Saralasin) and were found to be linear (Fig. 8A), which suggests that conventional saturation kinetics apply to the mechanisms involved. This is compatible with the account given by Goldstein et al. (1968) of the kinetics of interaction of drugs with receptor sites. The 1 000
\\ \
to= 32 5 sec P~~~~~~~A
AE100 \
0'~~\ a>7
I-
\
B to= 15 2 sec
Q
C2t3= 13 0 sec
U)
'-00
C -
0 10 -
~~~~~~Dtj= 12'3 sec
E ts= 12;2 sec 10 20 0 30 40 50 60 Time after starting angiotensin 11-amide infusion
Fig. 5. The measurement of the half-life of angiotensin II-amide from the onset of the pressor response; examples taken from one rat. Blood pressure measurements (read at 5 see intervals following the start of the infusion) were converted to readings of (infusion rate minus 'equivalent infusion rate') as described in the Methods section. A, results following the start of an infusion of 1000 ng angiotensin II-amide kg-. min-'; B, 500 ng kg-l. min-; C, 300 ng kg-". min-; D, 100 ng kg-. min'; E, 30 ng kg-1. min-1.
intercept of this line on the y-axis indicates the maximum (theoretical) rate of decrease of blood pressure, were it possible to administer an infinitely large dose of inhibitor. This value was 75 + 7 mmHg. min-' (at oc in Fig. 8B) which shows that the response to angiotensin II-amide can disappear more than 100 % faster than it does following the arrest of an infusion of the drug. These observations show that the
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES
479 pressor response can change fast enough to indicate accurately alterations in the concentration of angiotensin II-amide Convolution integrals were calculated (Jacquez, 1972) and drawn by computer to show how the inferred concentration of angiotensin II-amide would vary if its concentration were to decay with a tj of 15 see and if the pressor response were to dissipate with a tj of 7 5, 10, 15 or 30 sec. Since the pressor response was found to dissipate twice as fast with the inhibitor Saralasin as it did following the arrest of an angiotensin II-amide infusion, the value of 7-5 sec is considered to represent the tj of the pressor response. It is seen in Fig. 9 that after a short delay of 14 sec, lines a and b become parallel and therefore indicate similar half-lives. Thus the observed dissipation rate of the pressor response (viz. 7-5 sec) is not slow enough to distort the TABLE 1. Circulating half-lives of renin, angiotensin II and angiotensin analogues in the rat Half-life Number of rats (sec) Method 16.2 + 1.3 8 (1) Angiotensin II (Asp-Ile5-angiotensin II) 16 15.2+ 1P1 (1) Angiotensin JI-amide 6 16.3+ 2.1 (2) (Asnl-Val5-angiotensin II) 5 15.1 + 1.3 (3) 10 14-0+ 10 (1) Angiotensin III (des-Asp'-angiotensin II) 6 384.0± 3-0 (4) Saralasin (Sarl-Ala8-angiotensin II) 4 (3) 181.0± 25.0 Renin The t s were determined from (1) decay of pressor activity following continuous infusions, (2) onset ofpressor activity with continuous infusions, (3) decay of pressor activity using the simplified injection technique, (4) decay of depressor activity using the simplified injection technique.
measured tj of angiotensin II-amide. Only if the pressor response had a half-life greater than the half-life of angiotensin II-amide would it cause serious error (Fig. 9, curve E). Fig. 10 shows the average blood pressure responses during 10 min infusions of six different doses of angiotensin II-amide (100-8000 ng. kg-'. min-'), with observations from six animals at each dose. A substantial degree of tachyphylaxis only occurred at the highest dose (8000 ng . kg-'. min-'), slight tachyphylaxis was observed with 2000 ng. kg-'. min-', but not with lower doses like those that were used for determining the ti. Blood samples (0.1 ml.) were collected from six rats, 1 min and 9 min after starting an infusion of 8000 ng angiotensin II-amide kg-'. min-' and were injected into bioassay rats. At 1 min the rats receiving the angiotensin II-amide infusion showed a blood pressure rise of 89 + 8 mmHg above the pre-infusion level; 0-1 ml. blood collected at this time caused a 43 + 5 mmHg deflexion in the bio-assay rats. At 9 min the blood pressure of the angiotensin It-amide-infused rats had fallen to 35 + 8mmHg above the pre-infusion level (P < 0-01, Wilcoxon test; comparison with 1 min values); 0-1 ml. blood collected at this time caused a 43-1 + 6-2 mmHg deflexion in the bio-assay rats, which was not significantly different from the value obtained with the 1 min blood samples (difference between deflexions = 0'2 + 0 7 mmHg; P > 0 05,
480 S. A. M. A. AL-MERANI AND OTHERS Wilcoxon test). These observations indicate that during sustained angiotensiD IIamide infusion, there is no accumulation of any products of catabolism (e.g. peptide fragments) capable of inhibiting the pressor activity of the angiotensin II-amide. These observations also show that when tachyphylaxis occurs, the pressor activity in the blood is maintained. 300 _
A
n
(n Q
D, E._
o0
L
Q 0
200 C-
,.-
I
E 0E 10
To
C
0 B
0
in
1-
C.,
20
0 500 100 Infusion rate of angiotensin 11-amide (ng kg-'
1000
min-')
Fig. 6A, the initial rate of increase of blood pressure versus the infusion rate of angiotensin II-amide. B, the dependence of the measured half-life upon the infusion rate at which the measurement was taken. These values were determined during the onset of angiotensin II-amide action (n 6 rats). With the lower doses of angiotensin II-amide, the rate of blood pressure increase was proportional to the dose used and the half-life was independent of the dose. =
at various doses into eighteen rats and the Angiotensin II-amide was infused 0-1 blood ml. of pressor activity samples was measured using bio-assay rats, and expressed as ng angiotensin II-amide/ml. plasma. These measured concentrations were corrected for catabolism during the time taken for transfer of the blood samples. To test that angiotensin-like chemicals were the only pressor substances present, blood samples were also assayed in bioassay rats receiving 1 fig/min Sar'-Leu8i.v.
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES 41 angiotensin II (which is a potent inhibitor of the pressor response to angiotensin IIamide; Fig. 12): these assay rats showed no blood pressure change on injection of 0.1 ml. blood from the angiotensin JI-amide-infused rats (the deflexion was 0+ o mmHg; n = 6 observations), but showed a 15+5 mmHg deflexion (n = 6) with 20 ng noradrenaline, and 23 + 5 mmHg deflexion (n = 6) when 200 ng tyramine was injected. Stop angiotensin
Stop angiotensin Il-amide infusion
X
II- amide infusion
A
B
mmHg mir 5 pg Saralasin 160
E
D
s
C
1m
130-I
I
\
E
-..
-
_r
AA
\
" -16 mmHg min-' 20 pig Saralasin F
-20 mmHg min-' \J 50 pig Saralasin
_w vf
-29 mmHIg rrnin-1
100
\
G
-25 mmHg min-' 10 lig Saralasin t
.
pg Saralasin
I
-61 mmHg mirn-'
H
-51 mmHg min-1 x200 pig Saralasin
-64 mmHg min-'
Fig. 7. Typical blood pressure recordings from one rat. A, B: the initial rate of fall of blood pressure when an angiotensin II-amide infusion was stopped. C-H: the initial rate of fall of blood pressure when various doses of Saralasin were injected during a continuous infusion of angiotensin II-amide. The angiotensin II-amide was infused at 300 ng. kg-'. min-'.
The line obtained by plotting the rise in angiotensin II-amide concentration versus the infusion rate did not pass through the origin (Fig. 11). This discrepancy, which can also be observed in the published data of Caravaggi et al. (1976) and Cain et al. (1970) may arise from the suppression of endogenous angiotensin production caused by the infusion. A regression line was calculated to fit these data (r = + 0-92; Fig. 11), with slope 21 x 10-3 (ng angiotensin II-amide/ml. arterial plasma)/(ng infused. kg-1. min-'). The t4 may then be calculated using eqn. (1). Hodge et al. (1967) have suggested that the distribution volume (V) is equal to the plasma volume; this is 36 ml./kg body wt. for the rat (Willson, Chapman & Munday, 1976). These data I6
PHY
278
482 S. A. M. A. AL-MERANI AND OTHERS yield a tj of 30 see for angiotensin II-amide. We take this to represent good agreement with the measurements described above, since eqn. (1) requires that the mean concentration of angiotensin II-amide be calculated for the whole distribution volume. Angiotensin however, is unevenly distributed, having a significantly higher concentration in arterial plasma than in venous plasma (Hodge et al. 1967). Thus the value of -0050T
A
I E 0,
I E
0-025
-01
02
01
0 //s (/Ig -1)
100 r
B
C
100
-
-
-~~~~~~~ E I
E2
-I- P
J. Phy8&l. (1978), 278, pp. 471-490 With 12 text-figure8 Printed in Great Britain
THE HALF-LIVES OF ANGIOTENSIN II, ANGIOTENSIN II-AMIDE, ANGIOTENSIN III, SAR1-ALA8-ANGIOTENSIN II AND RENIN IN THE CIRCULATORY SYSTEM OF THE RAT
BY S. A. M. A. AL-MERANI, D. P. BROOKS, B. J. CHAPMAN AND K. A. MUNDAY and Pharmacology, School of Biochemical and Physiology From the Department of
Physiological Sciences, University of Southampton, Southampton S09 3TU (Received 3 November 1977) SUMMARY
1. Methods are described for estimating the half-life of angiotensin analogues and renin in the rat, from the time course of the blood pressure changes they evoke. 2. The following half-life values were measured: angiotensin II, 16 + 1 see; angiotensin III, 14+ 1 see; angiotensin II-amide, 15±+1 see; Sarl-Ala8-angiotensin II, 6-4 + 0-6 min; renin, 3 0 + 0 4 min. The apparent distribution volume of angiotensin was found to be 18 ml./kg body wt. 3. It is inferred that the Asp' residue does not reduce the rate of angiotenrsin II catabolism, but that substitution of this residue by sarcosine may inhibit catabolism while substitution by asparagine has no effect. 4. Five experimental criteria were identified which indicate that these methods give reliable estimates of the half-life. It is suggested that these results are more accurate than most previous half-life estimates. 5. When tachyphylaxis to angiotensin II-amide occurs, the pressor activity of the plasma is not reduced. INTRODUCTION
Studies of the half-life (t4) of angiotensin II have produced some conflicting results and very little is known of the half-lives of angiotensin analogues. Hodge, Ng & Vane (1967) measured the capacity of various organs to remove angiotensin II from the circulation, and calculated a t1 of 15 see in the dog. Wolf, Mendlowitz, Gitlow & Naftchi (1962) measured the t1 of [131I]angiotensin II as between 10 and 37 hr in man, although this probably represents the biological half-life of the 131I after break-down of the angiotensin molecule. Corcoran, Kohlstaedt & Page (1941) assessed the tj of a crude angiotensin preparation to be 20-25 min. Cain, Catt, Coughlan & Blair-West (1970), and Boyd, Landon & Peart (1969) found a tj of about 1 min for angiotensin II; but these workers used immunoassay techniques which may have detected inactive peptides formed by angiotensin hydrolysis, and hence may have underestimated the inactivation of the hormone. Regoli, Rioux, Park & Choi (1974) found that Sarl-angiotensin II has a reduced rate of catabolism in vitro and concluded that the greater pressor activity of the molecule may well result from a longer half-life in vivo. Similarly, Peach (1977) has sug-
S. A. M. A. AL-MERANI AND OTHERS gested that the in vivo tj of des-Aspl-angiotensin II may be 2-3 times shorter than that of angiotensin II, since it is catabolized faster in vitro. There are therefore important reasons for determining accurately the half-lives of angiotensin II and related compounds in the circulatory system, especially since it is possible to relate such measurements to studies of the in vivo actions of these substances. We wanted to measure these half-lives in the rat, but the rat is too small to permit the withdrawal of serial blood samples for angiotensin II assay and also there are no assay methods for some angiotensin analogues. We have therefore measured the half-lives of these various substances by studying the time course of the pressor response when a continuous infusion of each substance was initiated or arrested. The half-life of a hormone is usually determined by measuring the decrease in hormone concentration when an infusion of the hormones is stopped. However, it is not essential to measure the hormone concentration in absolute units; it is only required to find the time taken for the concentration, however expressed, to change by a factor of 0.5. It is established that the rise in plasma concentration of angiotensin II is proportional to the rate at which it is infused; any plasma concentration can therefore be quantified in terms of the infusion rate which could generate that concentration. We have assessed the changes in hormone concentration as follows: blood pressure readings were taken at regular intervals and interpreted as 'equivalent infusion rates' by reference to a calibration graph of blood pressure vs. infusion rate. Some of this work has been included in a brief report elsewhere (Brooks, Chapman & Munday, 1977). 472
METHODS
250 g female, Wistar albino rats (given 0-9 % saline in place of their drinking water for at least 10 days) were anaesthetised with sodium pentobarbitone and cannulae were placed in the trachea, one or both femoral veins (for infusion and/or injection of drugs) and in a femoral artery (for the measurement of blood pressure using an S.E. Labs. blood pressure transducer and a Servoscribe, model 45, potentiometric recorder). The following substances were dissolved in 0 9 % saline and either injected i.v. in 0 1-0 2 ml. or infused in 1 ml./hr at a variety of doses: angiotensin II-amide, i.e. Asn.-Valk-angiotensin II (Hypertensin, Ciba), 3-17,000 ng. kg-l. min'; angiotensin II, i.e. Asp'-Ile6-angiotensin II (Digby Chemical Company), 3-1000 ng. kg-'. min'; angiotensin III, i.e. des-Asp'-angiotensin II (D.C.C.), 20-4000 ng.kg-1.min-'; Saralasin, i.e. Sarl-Ala8-angiotensin II (D.C.C.) 5-200jg; Sarl-Leu8-angiotensin II (D.C.C.) 1 ,ug.kg-1.min-1; tyramine (Sigma) 200 jug; noradrenaline (Sigma) 20 jug; and pig renin (very kindly prepared by B. Rowe and A. R. Noble using the method of Haas, Goldblatt, Lewis & Gipson, 1972). The various doses of any chemical were applied in random sequence. Estimation of the ti values. Ginsberg (1968) and Goldstein, Aronow & Kalman (1968) have described the relationship between the infusion rate of a hormone (I), the volume (V) in which the hormone is distributed within the body, the t4, and the plasma concentration of the hormone at equlibrium (C): I~ (1) 0.693. V Since the 4 and V normally remain constant in any one animal, C is proportional to I; this has been verified in the case of angiotensin (see below). Thus any given infusion of angiotensin, I, will give rise to an equilibrium concentration C of the hormone and a pressor response R'. If the hormone is infused at 2 x I then the concentration of hormone at equilibrium will become 2 x C' and this will give rise to the pressor response R'. This pressor response R' will not necessarily be equal to 2 x R'. If this infusion is stopped, the concentration of hormone will decrease exponentially and the pressor response will dissipate at a rate determined by the disappearance rate of the
e=
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES
473
hormone (evidence in support of this is given below). When the pressor response has fallen to R' then the concentration of hormone must have fallen from 2C to C and the time taken must be equal to the half-life of the hormone. This principle was extended by measuring the magnitude of the pressor response at 5 see intervals after stopping the infusion. These responses were related to 'equivalent infusion rates' (and thus related to the concentration of hormone, since this is proportional to an infusion rate) by reference to a graph of equilibrium pressor response verbus infusion rate. Graphs of log 'equivalent infusion rate' versus time were constructed and the half lives determined from the slope of the resulting straight lines; ij = log 2/slope. For some experiments this basic method was further simplified by constructing graphs of (the peak pressor response) verseu (log dose) for single injections of the hormones; the time course of the decay of any response was then studied as described above. This is referred to as the simplified injection procedure. Goldstein et al. (1968) have shown that when an infusion of a hormone is started, the concentration of the hormone increases towards the equilibrium (plateau) concentration with a shift halftime that is equal to the half-life of the hormone. In some experiments the time course of the onset of the pressor response was therefore studied. The blood pressure was measured at five second intervals following the start of an infusion and graphs were plotted to show log (the infusion rate minus the 'equivalent infusion rate') versus time. The shift half-time, i.e. the tj of the hormone, was calculated from the slope of the resulting straight line, The half-life of Saralasin, which lacks pressor activity but is a potent inhibitor of angiotensin II action, was investigated as follows. 300 ng. kg-'. min- angiotensin II was continuously infused into each rat to give a constant rise in blood pressure, and various doses of the inhibitor (5200 jog) were injected i.v. in random sequences. The blood pressure falls due to the inhibitor were recorded and calibration graphs were constructed showing depressor response vernue log dose. From the recordings of the return of the blood pressure following the point of maximum inhibitory action of the Saralasin, graphs of log 'equivalent dose' verbu. time were constructed and the half-life of the inhibitor was determined. Bio-a88ay of pre88or activity in blood. Arterial blood samples (0-1 ml.) were taken from rats receiving 0-17-5 jug angiotensin II-amide kg-".min-' and injected I.v. into ganglion-blocked (pentolinium tartrate, 10 mg/kg) bio-assay rats (Peart, 1955). In some cases the pressor activity of these blood samples was assayed in terms of angiotensin II-amide concentration by bracketing with standard doses (Peart, 1955). The measured concentrations of angiotensin II-amide were corrected for catabolism during the period of transfer, by adding known amounts of angiotensin II-amide to blood samples and assaying the resulting pressor activity. An antagonist of angiotensin II pressor activity (Sarl-Leu8-angiotensin II) was infused at 1 #g/min i.v. into some bio-assay rats to give a preparation capable of detecting non-angiotensin pressor activity. All results are quoted as the mean ± 1 S.E. of the mean. The methods described by Colquhoun (1971) were used to evaluate the results statistically.
RESULTS
The tj of angiotensin II-amide Figs. 1 and 2 show the blood pressure recordings and the calibration graph of (plateau pressor response) versus (log infusion rate) obtained from one rat. The infusions were given for 2-5 min and were applied in random sequence but have been edited as shown. The decays of the pressor responses were studied for the three highest doses and used to construct the lines shown in Fig. 3. The average tt of angiotensin II-amide measured in this rat was 150O sec. The t1 of angiotensin II-amide was thus found to be 15-2 + 1-1 sec in sixteen rats. The measured tj was found to be the same for decays following a wide range of infusion rates (Fig. 4). These results show that the pressor responses were able to dissipate at very fast rates following high doses of the drug and that the initial,
474 S. A. M. A. AL-MERANI AND OTHERS steepest rate of decrease of the blood pressure, was proportional to the dose of angiotensin II-amide studied. There was usually a short time delay between switching off the infusion of the hormone and the start of the downsweep of the pressure recording. This delay, which was quantified by extrapolating the linear portion of the decay line as shown in Fig. 3, was found to be 10-4 + 0 7 seconds (n = 15). This delay was generally independent of the dose administered, and may have been due to the time taken for blood to travel between the site of infusion, the femoral vein, and the site of drug action. There may also be a time lay between the disappearance of the hormone and the reaction of the target tissue (see below). Dose of angiotensin lI-amide 3 ng. kg-'. min' 30 30 10
*6)
(mmHg)
-1110
I
_
Rise inB.P.at plateau
Rise= 6 mmHg
100
126
300
-1000
is
____
__
_15
T~~~~~~~~~~~~4
I~~~~~~
Fig. 1. Arterial blood pressure recordings obtained from a typical rat before, during and following various i.v. infusions of angiotensin II-amide. The speed of the chart recorder was increased at the points marked *, to permit detailed study of the decay curves.
The shift half-time was also determined from the onset of the pressor responses produced when various angiotensin II-amide infusions were switched on (Fig. 5). The shift half-time is the time for the concentration of a hormone to move 50% of the way towards its equilibrium concentration, when an infusion is started or changed. Goldstein et al. (1968) have shown theoretically and empirically that the upsweep of the concentration is a mirror-image of the disappearance of hormone when an infusion is switched off. The shift half-time is therefore equal to the tj for the destruction of the hormone. It was only possible to consider the initial part of the rise in blood pressure as the calibration curves did not have sufficient precision to permit evaluation of changes close to the plateau. The initial rate of increase of blood pressure was proportional to the dose of angiotensin II-amide used when the lower doses were infused; but when the two higher doses were used, the rate of increase of blood pressure was no longer proportional to the infusion rate (Fig. 6A). Similarly the
HALF-LIVES OF REIINJN AND ANGIOTENSIN ANALOGUES 475 measured half-life (i.e. the shift half-time) was independent of the infusion rate at the lower doses, but was observed to be prolonged during the infusion of 1000 ng. kg-1. min-' and to be slightly prolonged during the infusion of 500 ng. kg-1. min(see Figs. 5 and 6B). These observations are interpreted as indicating that the concentration of angiotensin II-amide follows a time course with a constant shift halftime, but that the rate of onset of the pressor response is limited by a maximum rate
60r-
40 0
I
E E C
U, .
_
20-
n v
0
100 10 Infusion rate of angiotensin 11 - amide (ng kg-1 min')
1000
Fig. 2. Calibration curve of the rise in B.P. vergu8 the infusion rate of angiotensin IIamide (taken from Fig. 1). The points were measured at the plateaux of the blood pressure responses.
of reaction of the target tissue (e.g. a maximum rate of contraction of the vascular smooth muscle). The results for the lower three doses were averaged for each animal. The over-all mean tj of angiotensin II-amide in six rats was then calculated and found to be 16-3 + 2 1 sec. This value was not significantly different from that calculated for the disappearance of the angiotensin II-amide (P > 0 05, Wilcoxon test). The half-life of angiotensin JI-amide was also determined by the simplified injection procedure (see Methods section) as 15-1 + 1t3 sees (n = 5 rats). This value is very similar to that obtained following constant infusions of the hormone (P > 0.05, Wilcoxon test), which indicates that the simplified injection procedure can give the same half-life results as does stopping a continuous infusion.
.' \t ~ ~ ~Trace6
S. A. M. A. AL-MERANI AND OTHERS
476
The half-life of natural angiotensin II and angiotensin III The half-lives were also determined for the disappearance of various analogues of angiotensin, which also have pressor activity: these were all found to have half-lives very similar to that of angiotensin II-amide (Table 1). 1000
From
Fig. 1
E
\U
6
Time lag
Measured,
t; (sec)
Trace 5
135
\Tae4
1-
100
15 *
30
-
Cr
10 50 10 20 30 40 60 Time after discontinuing angiotensin 11-amide infusion (sec)
Fig. 3. The exponential decrease in angiotensin II-amide concentrations, when various i.v. infusions were discontinued. Examples from one rat, taken from curves 4, 5, and 6 of Fig. 1. See Methods section for an explanation of how these graphs were constructed.
The half-life of rein The half-life of renin was determined by the simplified injection procedure and was found to be 3*02 + 0*41 min in four rats (Table 1). The half-life of Saralasin The half-life of Saralasin was found to be 6-43 ± 0*57 min in six rats; the modified procedure described in the Methods section was used.
Tests of the validity of the methods used. The initial rate of decrease of the blood pressure was determined for a variety of experimental conditions (Figs. 4, 7). When an infusion of 300 ng. kg-. min-' angiotensin JI-amide was discontinued the blood pressure initially decreased at 37*4 +
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES 477 2*6 mmHg. min-1 in seventeen rats (Fig. 8C). When an i.v. infusion of 25 ,ag.kg-1. min- acetylcholine was given to animals receiving an infusion of 300 ng . kg-.. mi r1 angiotensin II-amide, the blood pressure initially decreased at 1470 + 290 mmHg. min- (n = 4 rats) which is significantly greater than the rate of decrease following the arrest of the angiotensin II-amide infusion (P < 0 01, Wilcoxon test). CL 0. O.
100r
A
4,
: E 0)
tcX 0
(5)
E
a
(U' E
(11
00.C CD
E
C
30
0
0a)
Mean tj
0
-
15-2+1-1
0
300 1000 50 100 Dose of angiotensin 11-amide studied (ng kg-'
sec
4000
min-')
Fig. 4A, the initial rate of decrease of blood pressure versus the infusion rate of angiotensin II-amide studied. B, the dependence of the measured half-life on the infusion rate at which the measurement was obtained. (These graphs are plotted on log scales to accommodate the wide range of doses used; the numbers of animals are shown in parentheses.)
The initial rates of decrease of blood pressure were also measured following various 1.v. injections of Saralasin in animals receiving a sustained infusion of 300 ng.kg-'. min- angiotensin II-amide (Fig. 7). The rate of decrease of blood pressure following the highest dose of inhibitor (200 jug) was 70 + 11 mmHg. min' (n = 6 rats). This was significantly faster than the decrease following the arrest of an angiotensin II-amide infusion (P < 0*01, Wilcoxon test). Furthermore, five of the doses of Saralasin caused
478 S. A. M. A. AL-MERANI AND OTHERS the blood pressure to decrease faster than did stopping the angiotensin II-amide infusion (compare Fig. 8B, C). Graphs were plotted of the reciprocal of (the initial rate of decrease in blood pressure) versus the reciprocal of (the dose of Saralasin) and were found to be linear (Fig. 8A), which suggests that conventional saturation kinetics apply to the mechanisms involved. This is compatible with the account given by Goldstein et al. (1968) of the kinetics of interaction of drugs with receptor sites. The 1 000
\\ \
to= 32 5 sec P~~~~~~~A
AE100 \
0'~~\ a>7
I-
\
B to= 15 2 sec
Q
C2t3= 13 0 sec
U)
'-00
C -
0 10 -
~~~~~~Dtj= 12'3 sec
E ts= 12;2 sec 10 20 0 30 40 50 60 Time after starting angiotensin 11-amide infusion
Fig. 5. The measurement of the half-life of angiotensin II-amide from the onset of the pressor response; examples taken from one rat. Blood pressure measurements (read at 5 see intervals following the start of the infusion) were converted to readings of (infusion rate minus 'equivalent infusion rate') as described in the Methods section. A, results following the start of an infusion of 1000 ng angiotensin II-amide kg-. min-'; B, 500 ng kg-l. min-; C, 300 ng kg-". min-; D, 100 ng kg-. min'; E, 30 ng kg-1. min-1.
intercept of this line on the y-axis indicates the maximum (theoretical) rate of decrease of blood pressure, were it possible to administer an infinitely large dose of inhibitor. This value was 75 + 7 mmHg. min-' (at oc in Fig. 8B) which shows that the response to angiotensin II-amide can disappear more than 100 % faster than it does following the arrest of an infusion of the drug. These observations show that the
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES
479 pressor response can change fast enough to indicate accurately alterations in the concentration of angiotensin II-amide Convolution integrals were calculated (Jacquez, 1972) and drawn by computer to show how the inferred concentration of angiotensin II-amide would vary if its concentration were to decay with a tj of 15 see and if the pressor response were to dissipate with a tj of 7 5, 10, 15 or 30 sec. Since the pressor response was found to dissipate twice as fast with the inhibitor Saralasin as it did following the arrest of an angiotensin II-amide infusion, the value of 7-5 sec is considered to represent the tj of the pressor response. It is seen in Fig. 9 that after a short delay of 14 sec, lines a and b become parallel and therefore indicate similar half-lives. Thus the observed dissipation rate of the pressor response (viz. 7-5 sec) is not slow enough to distort the TABLE 1. Circulating half-lives of renin, angiotensin II and angiotensin analogues in the rat Half-life Number of rats (sec) Method 16.2 + 1.3 8 (1) Angiotensin II (Asp-Ile5-angiotensin II) 16 15.2+ 1P1 (1) Angiotensin JI-amide 6 16.3+ 2.1 (2) (Asnl-Val5-angiotensin II) 5 15.1 + 1.3 (3) 10 14-0+ 10 (1) Angiotensin III (des-Asp'-angiotensin II) 6 384.0± 3-0 (4) Saralasin (Sarl-Ala8-angiotensin II) 4 (3) 181.0± 25.0 Renin The t s were determined from (1) decay of pressor activity following continuous infusions, (2) onset ofpressor activity with continuous infusions, (3) decay of pressor activity using the simplified injection technique, (4) decay of depressor activity using the simplified injection technique.
measured tj of angiotensin II-amide. Only if the pressor response had a half-life greater than the half-life of angiotensin II-amide would it cause serious error (Fig. 9, curve E). Fig. 10 shows the average blood pressure responses during 10 min infusions of six different doses of angiotensin II-amide (100-8000 ng. kg-'. min-'), with observations from six animals at each dose. A substantial degree of tachyphylaxis only occurred at the highest dose (8000 ng . kg-'. min-'), slight tachyphylaxis was observed with 2000 ng. kg-'. min-', but not with lower doses like those that were used for determining the ti. Blood samples (0.1 ml.) were collected from six rats, 1 min and 9 min after starting an infusion of 8000 ng angiotensin II-amide kg-'. min-' and were injected into bioassay rats. At 1 min the rats receiving the angiotensin II-amide infusion showed a blood pressure rise of 89 + 8 mmHg above the pre-infusion level; 0-1 ml. blood collected at this time caused a 43 + 5 mmHg deflexion in the bio-assay rats. At 9 min the blood pressure of the angiotensin It-amide-infused rats had fallen to 35 + 8mmHg above the pre-infusion level (P < 0-01, Wilcoxon test; comparison with 1 min values); 0-1 ml. blood collected at this time caused a 43-1 + 6-2 mmHg deflexion in the bio-assay rats, which was not significantly different from the value obtained with the 1 min blood samples (difference between deflexions = 0'2 + 0 7 mmHg; P > 0 05,
480 S. A. M. A. AL-MERANI AND OTHERS Wilcoxon test). These observations indicate that during sustained angiotensiD IIamide infusion, there is no accumulation of any products of catabolism (e.g. peptide fragments) capable of inhibiting the pressor activity of the angiotensin II-amide. These observations also show that when tachyphylaxis occurs, the pressor activity in the blood is maintained. 300 _
A
n
(n Q
D, E._
o0
L
Q 0
200 C-
,.-
I
E 0E 10
To
C
0 B
0
in
1-
C.,
20
0 500 100 Infusion rate of angiotensin 11-amide (ng kg-'
1000
min-')
Fig. 6A, the initial rate of increase of blood pressure versus the infusion rate of angiotensin II-amide. B, the dependence of the measured half-life upon the infusion rate at which the measurement was taken. These values were determined during the onset of angiotensin II-amide action (n 6 rats). With the lower doses of angiotensin II-amide, the rate of blood pressure increase was proportional to the dose used and the half-life was independent of the dose. =
at various doses into eighteen rats and the Angiotensin II-amide was infused 0-1 blood ml. of pressor activity samples was measured using bio-assay rats, and expressed as ng angiotensin II-amide/ml. plasma. These measured concentrations were corrected for catabolism during the time taken for transfer of the blood samples. To test that angiotensin-like chemicals were the only pressor substances present, blood samples were also assayed in bioassay rats receiving 1 fig/min Sar'-Leu8i.v.
HALF-LIVES OF RENIN AND ANGIOTENSIN ANALOGUES 41 angiotensin II (which is a potent inhibitor of the pressor response to angiotensin IIamide; Fig. 12): these assay rats showed no blood pressure change on injection of 0.1 ml. blood from the angiotensin JI-amide-infused rats (the deflexion was 0+ o mmHg; n = 6 observations), but showed a 15+5 mmHg deflexion (n = 6) with 20 ng noradrenaline, and 23 + 5 mmHg deflexion (n = 6) when 200 ng tyramine was injected. Stop angiotensin
Stop angiotensin Il-amide infusion
X
II- amide infusion
A
B
mmHg mir 5 pg Saralasin 160
E
D
s
C
1m
130-I
I
\
E
-..
-
_r
AA
\
" -16 mmHg min-' 20 pig Saralasin F
-20 mmHg min-' \J 50 pig Saralasin
_w vf
-29 mmHIg rrnin-1
100
\
G
-25 mmHg min-' 10 lig Saralasin t
.
pg Saralasin
I
-61 mmHg mirn-'
H
-51 mmHg min-1 x200 pig Saralasin
-64 mmHg min-'
Fig. 7. Typical blood pressure recordings from one rat. A, B: the initial rate of fall of blood pressure when an angiotensin II-amide infusion was stopped. C-H: the initial rate of fall of blood pressure when various doses of Saralasin were injected during a continuous infusion of angiotensin II-amide. The angiotensin II-amide was infused at 300 ng. kg-'. min-'.
The line obtained by plotting the rise in angiotensin II-amide concentration versus the infusion rate did not pass through the origin (Fig. 11). This discrepancy, which can also be observed in the published data of Caravaggi et al. (1976) and Cain et al. (1970) may arise from the suppression of endogenous angiotensin production caused by the infusion. A regression line was calculated to fit these data (r = + 0-92; Fig. 11), with slope 21 x 10-3 (ng angiotensin II-amide/ml. arterial plasma)/(ng infused. kg-1. min-'). The t4 may then be calculated using eqn. (1). Hodge et al. (1967) have suggested that the distribution volume (V) is equal to the plasma volume; this is 36 ml./kg body wt. for the rat (Willson, Chapman & Munday, 1976). These data I6
PHY
278
482 S. A. M. A. AL-MERANI AND OTHERS yield a tj of 30 see for angiotensin II-amide. We take this to represent good agreement with the measurements described above, since eqn. (1) requires that the mean concentration of angiotensin II-amide be calculated for the whole distribution volume. Angiotensin however, is unevenly distributed, having a significantly higher concentration in arterial plasma than in venous plasma (Hodge et al. 1967). Thus the value of -0050T
A
I E 0,
I E
0-025
-01
02
01
0 //s (/Ig -1)
100 r
B
C
100
-
-
-~~~~~~~ E I
E2
-I- P
